Synthesis and Temperature-Responsiveness of Poly(ethylene glycol

Chapter 5

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Synthesis and Temperature-Responsiveness of Poly(ethylene glycol)-like Biodegradable Poly(ether-ester)s Yuichi Ohya,*,1,2 Akihiro Takahashi,2 Hiroki Takaishi,1 and Akinori Kuzuya1,2 1Department

of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan 2Organization for Research and Development of Innovative Science and Technology (ORDIST), Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan *E-mail: [email protected]

Poly(ethylene glycol) (PEG) is widely used as a biomedical material because it possesses the highest level of biocompatibility among synthetic polymers. However, PEG is water-soluble and non-biodegradable. To provide biodegradable water-insoluble bulk materials possessing the biocompatibility of PEG, water-insoluble poly(ether-ester)s were synthesized by thiol-ene polyaddition of poly(ethylene glycol) diacrylates (PEGDA) and alkyl dithiols (ADTs). Among them, P(PEG-DDT) prepared from PEGDA and decanedithiol (DDT) film cast on a glass substrate possessed temperature-responsive changes only when immersed in water. The P(PEG-DDT) film was transparent in a water bath at 0 ºC, but become opaque after subsequent soaking in a hot water bath at 40–70 ºC. The mechanism was examined by measuring transmittance changes in response to temperature, differential colorimetry, and air contact angle of the film in water. Water-insoluble poly(ether-ester)s exhibiting temperature-responsiveness have potential, not only as new bulk-state biomaterials with PEG-like biocompatibility but

© 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

also as new environmentally friendly temperature-responsive materials.


Introduction Poly(ethylene glycol) (PEG) is an amphiphilic polymer that is soluble in common organic solvents and in water, and is one of the most popular polymers used in biomaterials. It is a non-toxic, non-immunogenic polymer, and can be eliminated through renal and hepatic pathways. In addition, PEG demonstrates the lowest level of interaction with proteins or cells among synthetic and natural polymers (1–3). These PEG characteristics permit its use in a wide variety of established and emerging applications in pharmaceutics to improve the pharmacokinetic properties of drugs and drug carriers (4–16). Chemical modification using PEG (PEGylation) is effective in prolonging blood circulation times and the half-life in the body of drugs and drug carriers by preventing protein absorption and entrapment by reticuloendothelial systems. However, PEG is not biodegradable. Yamaoka et al. conducted a detailed study on the distribution and tissue uptake of PEGs with different molecular weights after intravenous (i.v.) administration into mice (17). The renal clearance of PEGs decreased with an increase in molecular weight, with the most dramatic change occurring at approximately 30,000 Da. This indicates that high-molecular-weight (>30,000 Da) PEG is difficult to excrete from the body, despite its biocompatibility. To provide suitable implantable biomaterials for temporal use, polymers must have a molecular weight high enough to escape renal clearance until their function is fulfilled and then degrade in the body. Thus, polymers possessing both PEG-like biocompatibility and the ability to biodegrade under physiological conditions are the best candidates for temporal implant biomaterials. Several groups have reported biodegradable poly(ether-ester)s composed of functional amino acids and PEG (18, 19). Another report described the synthesis of an alternative copolymer of PEG and L-aspartic acid, poly(L-Asp-alt-PEG), through polycondensation of PEG and an L-aspartic acid derivative. The poly(L-Asp-alt-PEG) obtained with alkyl side chains possessed both biodegradability and temperature-responsiveness, but did not have a molecular weight high enough to prevent renal clearance (20). High-molecular-weight poly(ether-ester)s are difficult to obtain via polycondensation. Wang et al. reported the synthesis of biodegradable and temperature-responsive PEG analogs (DPEGs) by thiol-ene polyaddition of dithiols and poly(ethylene glycol) diacrylates (PEGDA) or di(meth)acrylates. In aqueous solution, the DPEGs had a lower critical solution temperature (LCST), and the response temperature could be adjusted by changing the type of dithiol and the molecular weight of the PEG derivative (21, 22). Temperature-responsive polymers often contain PEG because it undergoes temperature-dependent dehydration in aqueous solution (23–26). Previously, PEGs have been used as a water-soluble polymer, solubilizer, and hydrophilic brush for a substrate surface in aqueous solution. However, water-insoluble “bulk” materials having excellent biocompatibility are also important as implantable biomedical materials. The present study describes 94 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the preparation of water-insoluble “bulk” materials possessing PEG-like biocompatibility and degradation to low-molecular-weight compounds that could be excreted from the body by employing a poly(ether-ester) backbone containing PEGDA and alkyldithiols (ADT). Several types of water-insoluble poly(ether-ester)s with relatively short PEG chains and various length of alkyl chains were synthesized. The process accidentally revealed that cast films of the water-insoluble copolymers obtained possessed temperature-responsive transmittance changes in hot water. Therefore, the synthesis of PEG-like biodegradable poly(ether-ester)s and their temperature-responsive properties were investigated. Poly(PEG-alkyl dithiol)s [P(PEG-ADT)s] composed of low-molecularweight PEGs and alkyl chains of various length were prepared via thiol-ene polyaddition of PEGDA and α,ω-alkyl dithiols. The temperature-responsiveness of the P(PEG-ADT) was evaluated by transmittance changes, calorimetric analysis, and contact angle measurements. The temperature-responsiveness and PEG-based biocompatibility of these water-insoluble poly(ether-ester)s are expected to be useful biocompatible biodegradable biomaterials.

Experimental Materials 1,6-Hexanedithiol (HDT), 1,10-decanedithiol (DDT), and triethylamine were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). The PEGDA (molecular weight = 700 Da), 1,4-butanedithiol (BDT), and 1,8-octanedithiol (ODT) were purchased from Sigma-Aldrich (St. Louis, USA). 1,2-Ethanedithiol (EDT) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Water was purified using a reverse osmotic membrane method (Milli-Q). Other solvents and reagents were commercial grade and used without further purification. Measurements 1H-

and 13C-nuclear magnetic resonance (NMR) spectra were recorded on a JNM-GSX-400 (Jeol, 400 MHz) instrument using deuterated chloroform (CDCl3) as a solvent. The chemical shifts were calibrated against tetramethylsilane (TMS) and the CDCl3 solvent signal. The weight-average molecular weight (Mw), number-average molecular weights (Mn), and polydispersity indices (Mw/Mn) of the copolymers were determined by size exclusion chromatography (SEC) (column: TSKgel Multipore HXLM × 2, detector: RI). Measurements were obtained using dimethylformamide (DMF) as eluent at a flow rate of 1.0 mL/min at 40 ºC and a series of PEG standards. Transmittance changes of the copolymer films were measured using a V-650 UV-Vis spectrophotometer (Jasco). Thermal analysis of the copolymers was conducted using a differential scanning calorimeter (DSC-60, Shimadzu) with sealed aluminum pans. Contact angles of the films were recorded using a contact angle meter (Kyowa Interface Science, Co., Ltd., DM-550). 95 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Synthesis of P(PEG-ADT)s


A series of P(PEG-ADT)s having various alkyl chain lengths were synthesized via thiol-ene polyaddition according to Scheme 1. Typical procedures for preparation of P(PEG-DDT) (y = 10 in Scheme 1) are as follows. A mixture of PEGDA (896 mg, 1.28 mmol) and DDT (264 mg, 1.28 mmol) was dried in vacuo and dissolved in anhydrous DMSO (8 mL). Triethylamine (73 mg, 0.60 mmol) was added to the solution and stirred at room temperature (r.t.) for 36 h. Then the reaction mixture was poured into diethylether/methanol (10/1, v/v) and washed three times. The precipitate obtained was dried in vacuo at r.t. to give P(PEG-ADT). Yield: 72%.

Scheme 1. The synthesis of P(PEG-ADT)s. 1H NMR (400 MHz, CDCl3): δ (ppm) = 1.20-1.31 (br, -CH2CH2CH2-), 1.31-1.42 (br, -SCH2CH2CH2-), 1.52-1.62 (m, -SCH2CH2CH2-), 2.47-2.55 (t, -SCH2CH2CH2-), 2.59-2.81 (m, -OC(=O)CH2CH2S-), 3.55-3.68 (br, -OCH2CH2O-), 3.68-3.73 (t, -COOCH2CH2O-), 4.23-4.28 (t, -COOCH2CH2O-). 13C NMR (100 MHz, CDCl3): δ (ppm) = 26.7, 28.8, 29.1, 29.3, 29.4, 32.0, 34.7, 63.7, 69.0, 70.5, 70.6, 172.1. Other P(PEG-ADT)s, P(PEG-ODT) (y=8), P(PEG-HDT) (y=6), P(PEGBDT) (y=4), P(PEG-EDT) (y=2), were also synthesized by the similar procedures.

Temperature-Responsiveness of Copolymer Films Cast copolymer films were prepared using a spincoater (1HD7, Mikasa Co. Ltd.). The P(PEG-DDT) was dissolved at a concentration of 100 mg/mL in CHCl3, spin-coated onto a flat glass substrate, and dried in vacuo at r.t. to give a transparent film ca. 500 μm thick. The temperature-responsive behavior of P(PEG-DDT) film on a glass substrate was evaluated in the presence and absence of water. The P(PEG-DDT) film on a glass-substrate was placed in ice-cold water, and then moved into a water bath at various temperatures. The appearance of the films was observed by the naked eye, and the film transmittance at 500 nm was measured. Transmittance changes of the films on a glass plate in the air (without water) also were observed. 96 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Results and Discussion


Synthesis of the Copolymers The P(PEG-ADT)s containing alkyl chains of different lengths were successfully synthesized via thiol-ene polyaddition using PEGDA and ADTs according to Scheme 1. Results are shown in Table 1. The Mn, Mw, and Mw/Mn values were in the ranges of 22,000–35,000, 37,000–58,000, and 1.62–2.11, respectively, and yields were greater than 67%. A trend toward a higher degree of polymerization (DP) for longer alkyl chain ADTs was observed. Elution profiles of the P(PEG-ADT)s are shown in Figure 1, and suggest successful purifications. The polydispersity indices (Mw/Mn) were relatively high, but unimodal distributions were observed for all P(PEG-ADT)s. Typical examples of 1H-NMR spectra of P(PEG-DDT) and the starting materials, PEGDA and DDT, are shown in Figure 2. The acryl group signals from PEGDA at 5.8–6.6 ppm were not present in the P(PEG-DDT) spectrum, suggesting that the terminal acryl groups completely reacted. New peaks at 2.6–2.8 ppm also were observed and assigned to –S–CH2CH2–C(=O) (reaction product of the acryl group). Other P(PEG-ADT)s gave similar results. All of the P(PEG-ADT)s obtained were insoluble in water. The P(PEG-EDT), P(PEG-BDT), P(PEG-HDT), and P(PEG-ODT) were viscous pastes in the dry state at r.t. and the viscosity of the copolymer increased with of alkyl chain length. The P(PEG-DDT) was solid after drying. However, preparing self-standing films from P(PEG-ADT)s, including P(PEG-DDT), was not possible.

Table 1. Results of synthesis of P(PEG-ADT)s Name of P(PEG-ADT)

ya

Mn (Da)b

Mw (Da) b

Mw/Mn b

DP c

Yield (%)

P(PEG-EDT)

2

23,000

49,000

2.11

29

73

P(PEG-BDT)

4

22,000

37,000

1.70

26

69

P(PEG-HDT)

6

26,000

46,000

1.75

31

76

P(PEG-ODT)

8

27,000

44,000

1.62

31

67

P(PEG-DDT)

10

35,000

58,000

1.66

39

a

Number of alkyl chain of ADTs (y in Scheme 1). polymerization estimated from Mn.

b

Estimated by SEC.

72 c

97 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Degree of


Figure 1. SEC profiles for P(PEG-EDT), EDT (a), P(PEG-BDT), BDT (b), P(PEG-HDT), HDT (c), P(PEG-ODT), ODT (d), P(PEG-DDT), DDT (e), and PEGDA. (see color insert)



Figure 2. 1H-NMR spectra of P(PEG-DDT) (top), PEGDA (middle) and DDT (bottom) in CDCl3. (see color insert)

Temperature Responsiveness The P(PEG-DDT) was solid in dry state at r.t. and insoluble in water at 0–100 ºC, but exhibited temperature-responsiveness when placed in water. As shown in Figure 3, P(PEG-DDT) film cast on a glass-substrate was transparent at 0–100 ºC in air. In contrast, despite the transparency of P(PEG-DDT) film in water at 0 ºC, it become opaque after placement in a water bath at 40–70 ºC. The turbidity of the film at 70 ºC appeared to be slightly greater than that at 40 ºC. These results indicate that the P(PEG-DDT) film underwent a temperature-responsive phase transition only in the presence of water. The turbidity change may be caused by phase separation of the copolymer on hydration/dehydration of the PEG segments. In addition, film kept in water at 40 ºC for 25 min was still translucent, but turbidity was decreased slightly compared to that before incubation. The turbid P(PEGDDT) film created by placement in hot water became transparent after placement in an ice-cold water bath again. These results demonstrate that the temperatureresponsive transparency change was reversible.



Figure 3. Photographs of P(PEG-DDT) films in the air or water at various temperature and history.

The temperature-responsive transmittance changes of P(PEG-DDT) films also were investigated by UV-Vis spectrophotometry. Transmittance changes of P(PEG-DDT) films placed in an ice water bath and then transferred to warmer water were measured. Figure 4a shows plots of film transmittance vs. duration of time in the warm water bath. When P(PEG-DDT) film was transferred to a water bath at 10 ºC, almost no transmittance change was observed. However, the transmittance of the film decreased dramatically to 20% within 1 min after transfer into a water bath at 40 ºC. The transmittance decreased to approximately 10% within 30 sec when the film was placed in a water bath at 70 ºC. Interestingly, the decreased transmittance values recovered slowly upon continued incubation in the warm water bath. These results indicate that the films exhibited temperature-responsive transmittance changes in the presence of water, but the change was temporal. Figure 4b shows plots of minimum transmittance value and transmittance value after 23 min in the warm water bath vs. water bath incubation temperature. The minimum transmittance value decreased dramatically when water temperature was 20–50 ºC, with a transition point of approximately 35 ºC. These results suggest that temperature-triggered phase separation of the films occurred by dehydration of PEG segments of the copolymer, which was accelerated with an increase in incubation temperature. In addition, the dehydration-induced phase separation was temporal and gradually reached an equilibrium state at the incubation temperature.



Figure 4. a) Time course of transmittance change at 500 nm after temperature change of P(PEG-DDT) film in water. The films were soaked in ice-cooled water (0 ºC) and subsequently moved to 10 ºC (dotted black line), 40 ºC (dashed blue line), 70 ºC (solid red line) water bath. b) Plots of minimum transmittance (●) and transmittance after 23 min (▲) vs. incubation temperature change for P(PEGDA-DDT) film. (see color insert)

To investigate the phase transition behavior, calorimetric analysis of the films was conducted using DSC. Figure 5 shows DSC plots of P(PEG-DDT) in the presence and absence of water. The P(PEG-DDT) without water showed endothermic peaks corresponding to a glass-transition temperature (Tg) and melting temperature (Tm) of -61 and 6 ºC, respectively. In contrast, DSC of P(PEG-DDT) with water showed several new peaks in addition to Tg and Tm. The Tm overlapped a large free water peak. An endothermic peak from -40 to -20 ºC was attributed to eutectic composition between the PEG segment of P(PEG-DDT) and water. Reports have demonstrated that PEG shows an endothermic eutectic peak with water from - 40 to -8 ºC, with the temperature dependent on molecular weight (27–31). The endothermic shoulder peak observed from -10 to 0 ºC was attributed to melting of bound water (30, 31). In addition, a small broad endothermic peak was observed from 25 to 75 ºC, which was assigned to dehydration of P(PEG-DDT), because this peak was observed only in the presence of water.



Figure 5. DSC charts for P(PEG-DDT) in water (red), P(PEG-DDT) (blue), and water (black, dotted line) from -100ºC to 130 ºC. (see color insert) To investigate the change in surface properties upon the temperature change, the water contact angle and air contact angle in water were investigated. Figure 6 shows the time course of water contact angle in the air at 20 and 40 ºC. The contact angles decreased gradually from approximately 110º to approximately 65º in 2 min. No significant difference was observed between 20 and 40 ºC. A gradual decrease in water contact angle is typical behavior for hydrophilic-hydrophobic copolymers due to conformational changes in the polymer chains at the air-water interface. Therefore, this phenomenon was not likely to be closely related to the temperature-responsiveness of P(PEG-DDT). Figure 7 shows the time course of air bubble contact angle in water during the temperature change from 0 to 40 ºC. No obvious contact angle change was observed. Therefore, no meaningful information was obtained from the contact angle measurements, and the surface hydrophilicity change was not critical for the temperature-responsive transmittance changes of this polymer. Although the mechanism for the temperature-responsive transmittance change of this polymer remains unclear, the interaction of PEG with water causing processes such as dehydration must play a critical role.

Figure 6. Time course of water contact angle for P(PEG-DDT) at 20 ºC (○) and 40 ºC (◆). (see color insert) 102 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 7. Time course of air bubble contact angle for P(PEG-DDT) in water after the temperature change from 4 ºC to 40 ºC. The films were soaked in cooled water (4 ºC) and subsequently moved to 40 ºC water bath.

Conclusions P(PEG-ADT)s, including P(PEG-DDT), were prepared successfully by thiol-ene polyaddition of PEGDA and ADTs. The Mw values of the polymers obtained were greater than 37,000. The P(PEG-DDT) film cast on glass possessed interesting temperature-responsive behavior. The transparency of the film did not change upon heating in air. However, in a water bath, the transmittance of the P(PEG-DDT) film changed from transparent to translucent upon an increase in temperature. This phenomenon was reversible with another temperature change: the film returned to transparency on cooling. The P(PEG-DDT) showed a broad endothermic peak from 25 to 75 ºC due to dehydration of the PEG segments, which was related to the polymer’s temperature-responsive behavior. The mechanism for the temperature-responsive transmittance change remains unclear at present, but this water-insoluble, temperature-responsive poly(ether-ester) containing PEG possesses great potential, not only as a new bulk-state biomaterial with PEG-like biocompatibility and biodegradability but also as a temperature-responsive environmental-friendly material for new applications.

Acknowledgments This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 25282147, and 16H01854) from the Japan Society for the Promotion of Science (JSPS), and by the Kansai University Subsidy for Supporting Young Scholars, 2013 and Kansai University Outlay Support for Establishing Research Centers, 2016.

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Synthesis and Temperature-Responsiveness of Poly(ethylene glycol

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